Global focus towards reducing petroleum footprint has led to a significant interest in developing alternative methods to produce fuels from low-cost and renewable resources. Metabolic engineering has emerged as an enabling technology to this end, which directs modulation of metabolic pathways by using recombinant technologies to overproduce valuable products, including biofuels [4-7]. Alkenes, traditionally used as detergents, lubricating fluids and sanitizers [8], have the potential to serve as “drop-in” compatible hydrocarbon fuels because of their high energy content. In addition, as they are already predominant components of petroleum-based fuels [9, 10], they are compatible with the existing engine platform and fuel distribution systems. Therefore, there is a strong economic and environmental demand for the development of bio-alkenes, which could be low-cost and environmentally sustainable, through metabolic engineering strategies.
The fatty acid biosynthesis pathway is ideally suited to provide biofuel precursors because of the high energy content in the precursors, and these fatty acid precursors can be converted into alkenes via naturally occurring metabolic pathways [1, 11-14]. The first pathway involves a cytochrome P450 fatty acid decarboxylase—OleTJE from Jeotgalicoccus sp. ATCC 8456 which directly decarboxylates free fatty acids to terminal alkenes [1-3]. The second pathway employs a multi-domain polyketide synthase, found in the cyanobacterium Synechococcus sp. PCC 7002. This enzyme converts fatty acyl-ACP to terminal alkene via an elongation decarboxylation mechanism [11]. The third pathway produces long-chain internal alkenes (C24-C31) by a head-to-head condensation of two acyl-CoA (or-ACP) thioesters followed by several reduction steps in Micrococcus luteus [12] and Shewanella oneidensis [13, 14]. Among these three pathways, the one-step fatty acid decarboxylation pathway is highly advantageous for alkene biosynthesis for the following two reasons. Firstly, the fatty acid synthesis pathway is feedback-inhibited by fatty acyl-CoA/ACP [15, 16], a precursor of fatty acid-derived biofuels. This feedback inhibition could negatively affect the boosting of fatty acyl-CoA/ACP levels, and in turn the fatty acid-derived biofuel titers. Thus, using free fatty acids as biofuel precursors is more desirable compared with fatty acyl-CoA/ACP. Secondly, a one-step reaction from fatty acids to alkenes reduces intermediate metabolite losses and toxicity [17-19].
The well-studied industrial microorganism Saccharomyces cerevisiae offers a number of advantages [20-23] for producing fatty acid-derived products due to i) its ability to withstand lower temperatures, ii) immunity towards phage contaminations, iii) suitability in large-scale fermentation, iv) generally higher tolerance toward abiotic stresses, and v) extensive knowledge available about its fatty acid metabolism.